This project focusses on the regulation of the calcium (Ca) pump of the cardiac sarcoplasmic reticulum (SR) by its natural regulator, phospholamban (PLN). PLN is a 27 KDa protein that is phosphorylated by protein kinase A as a result of catecholamine induced stimulation of adenylate cyclase. Recent kinetic data indicate that maximal velocity of Ca uptake at saturating Ca2+ (V) is decreased by unphosphorylated PLN in addition to its well known effect on K. Our findings are consistent with an inhibition by PLN at least two steps in the catalytic cycle, namely, E2--E1 and E2P -> E2P+P(E2P decomposition). Our working model (hypothesis) ascribes PLN's effects on Ca2+ pump affinity and V on these two steps, respectively. The inhibitory effect of PLN appears to involve the membrane bilayer, at or close to the membrane surface. Phospholipid have previously been implicated as a requirement for E2P decomposition and may be involved in the regulation of this step by PLN. We propose to test this model in studies with the following specific aims. (1) TO extend our findings of effects of PLN on both apparent V and K of Ca uptake and their dissociation by different agents including effects of polyanions/calmodulin and palmitylcarnitine. (2) To test synthetic peptide analogs of PLN for effects on the kinetic parameters of Ca uptake and Ca-ATPase activities in SR vesicles. (3) To determine the effect of PLN on regulatory nucleotide binding by the Ca pump protein using 2,3-0- (2,4,6-trinitrophenyl)adenosine 5'-triphosphate, which will also be used to determine effects of PLN and PLN peptides on E2P decomposition. (4) To localize the site(s) of interaction of active PLN peptide analogs on the SR calcium pump protein in crosslinking studies. (5) To investigate the extent of interaction of active PLN-analog peptides with the membrane bilayer by use of fluorescence quenching and resonance energy transfer methodologies. The elusiveness of a thorough understanding of the interaction of PLN with the SR Ca pump is perhaps related to our incomplete knowledge of the energy transduction process itself. The proposed studies should provide new information in both of these areas as well as in basic cardiac physiology.